DESCRIPTION: The objective of this application is to conduct studies to understand mechanisms by which hypoxia followed by reoxygenation (a model of ischemia/reperfusion) causes tissue injury. The applicant hypothesizes that reoxygenation increases the activity of a novel cell membrane oxidase that results in the enhanced production of deleterious reactive oxygen species. The proposed studies will use various endothelial cells in culture to investigate the role of the membrane associated oxidase activity as well as xanthine oxidase and nitric oxide synthase in the generation of reactive oxygen species under conditions of hypoxia and reoxygenation. The specific aims are: 1) to investigate the presence of an NADPH oxidase in plasma membranes of endothelial cells by Northern and Western blot analysis; 2) to investigate the role of xanthine oxidase in the generation of intracellular reactive oxygen species and the mechanisms by which allopurinol and other antioxidants exert protective effects; and 3) to investigate the role of nitric oxide synthase in the pathogenesis of tissue injury by hypoxia and reoxygenation. During performance of the project, it is anticipated that Dr. Zulueta will become expert in facets of cellular biology and oxidant chemistry and will learn contemporary procedures for studies of cellular molecular biology and PCR technology.